The present invention relates generally to compositions and methods useful for inhibiting the multiplication of human immunodeficiency virus-1 (HIV-1) in infected patients, symptomatic or asymptomatic, and for attenuating HIV-1 multiplication during primary infection in previously uninfected subjects, thus minimizig progression to AIDS.
High plasma levels of human immunodeficiency virus type 1 (HIV-1) RNA are found during primary infection with HIV-1, the seroconversion illness, [C. Baumberger et al, AIDS, 7:(suppl 2):S59 (1993); M. S. Saag et al, Nature Med., 2:625 (1996)], after which they subside as the immune response controls the infection to a variable extent. Post seroconversion, lower but detectable levels of plasma HIV-1 RNA are present, and these levels rise with disease progression to again attain high levels at the AIDS stage [M. S. Saag et al, Nature Med., 2:265 (1996)]. Approximately 50% of subjects have a symptomatic illness at seroconversion [B. Tindall and D. A Cooper, AIDS, 5:1 (1991)] and symptomatic seroconversion is associated with an increased risk for the development of AIDS, probably because a severe primary illness is likely related to an early and extensive spread of HIV.
Inhibition of viral multiplication during the initial infection will likely reduce the subsequent development of chronic viremia leading to AIDS. Current medical practice, with administration of antiviral drugs for defined xe2x80x9cat riskxe2x80x9d situations, such as needle sticks with contaminated blood or pregnancy in HIV infected mothers, supports this concept.
Post seroconversion levels of HIV-1 RNA in plasma have proven to be the most powerful prognosticator of the likelihood of progression to AIDS [J. W. Mellors et al, Science, 272:1167 (1996); M. S. Saag et al, Nature Med., 2:265 (1996); R. W. Coombs et al, J. Inf. Dis., 174:704 (1996); S. L. Welles et al, J. Inf. Dis., 174:696(1990)]. Other measures of viral load, such as cellular RNA [K. Saksela et al, Proc. Natl. Acad. Sci. USA, 91:1104 (1994)] and cellular proviral DNA [T-H. Lee et al, J. Acq. Imm. Def. Syndromes, 7:381 (1994)] similarly establish the importance of the initial infection in establishing viral loads that determine future disease progression.
Thus, any intervention that inhibits HIV-1 infectivity during initial infection and/or lowers viral load post sero-conversion is likely to have a favorable influence on the eventual outcome, delaying or preventing progression to AIDS.
A variety of methods are now employed to treat patients infected with human immunodeficiency virus (HIV-1), including treatment with certain combinations of protease inhibitor drugs. Unfortunately, however, this type of treatment is associated with serious side effects in some patients. Alternatively, vaccines are under development for control of the spread of HIV-1 to uninfected humans. However, this effort has largely been directed to proteins of the virus, expressed on the surface of infected cells, which are recognized by cytotoxic T cells with elimination of the infected cells, while free virus is blocked and cleared by antibody to surface antigens of the virion. Limitations of this mode of vaccination are readily apparent for HIV-1, which has demonstrated a great diversity in immunogenic viral epitopes and rapid mutational variations that occur within and between individuals [B. D. Preston et al., Science, 242:1168 (1988); J. D. Roberts et al., Science, 242:1171 (1988); A. R. Meyerhans et al., Cell, 58:901 (1989); K. Kusumi et al., J. Virol., 66:875 (1992); B. A. Larder et al., Science, 243:1731 (1989); M. S. Sang et al., N. Engl. J. Med., 329:1065 (1993); M. A. Sande, et al., JAMA, 270:2583 (1993); M. Seligmann et al., Lancet, 343:871 (1994); G. Meyers et al., Human retroviruses and AIDS 1993, I-V. A compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N. Mex.]
Variation in strains of HIV-1 and frequent mutations of virion proteins have prevented successful application of conventional vaccine approaches [W. E. Paul, Cell, 82:177 (1995); J. E. Osborn, J. Acq. Imm. Def. Syndr. Hum. Retrovirol., 9:26 (1995)]. Mutation and selection of resistant variants is the central problem in developing a successful HIV-1 vaccine [M. D. Daniel et al., Science, 258:1938 (1992); N. L. Letvin, N. Engl. J. Med. 329:1400 (1993); M. Clerici et al., AIDS, 8:1391 (1994); S. M. Wolinsky et al, Science, 272:537 (1996)].
Other approaches to HIV-1 treatment have focused on the transactivating (tat) gene of HIV-1, which produces a protein (Tat) essential for transcription of the virus. The tat gene and its protein have been sequenced and examined for involvement in proposed treatments of HIV [see, e.g., U.S. Pat. No. 5,158,877; U.S. Pat. No. 5,238,882; U.S. Pat. No. 5,110,802; International Patent Application No. WO92/07871, published May 14, 1992; International Patent Application No. WO91/10453, published Jul. 25, 1991; International Patent Application No. WO91/09958, published Jul. 11, 1991; International Patent Application No. WO87/02989, published May 21, 1987]. Tat protein is released extracellularly, making it available to be taken up by other infected cells to enhance transcription of HIV-1 in the cells and to be taken up by noninfected cells, altering host cell gene activations and rendering the cells susceptible to infection by the virus. Uptake of Tat by cells is very strong, and has been reported as mediated by a short basic sequence of the protein [S. Fawell et al., Proc. Natl. Acad. Sci. USA, 91:664-668 (1994)].
International Patent Application No. WO92/14755, published Sep. 3, 1992, relates to the Tat protein and to the integrin cell surface receptor capable of binding to the Tat protein. Two Tat sequences that bind integrin are identified, which are the basic region or domain which is the dominant binding site for the integrin, having a peptide sequence of xe2x80x94Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Argxe2x80x94 [SEQ ID NO: 4], as well as xe2x80x94Gly-Arg-Gly-Asp-Ser-Proxe2x80x94 [SEQ ID NO: 5]. This specification demonstrates that a number of peptides corresponding to these Tat sequences and the corresponding integrins block in vitro cell binding to Tat coated plates, as do antibodies to the appropriate integrins. However, the specification also shows that these reagents do not block uptake of functional Tat by cells (see Example 9 in WO92/14755), thus nullifying the proposed mechanism of action for therapeutic benefit in HIV infection. The Tat sequences described in this international application are distinct from the peptide immunogens of the present invention.
Both monoclonal and polyclonal antibodies to Tat protein have been readily produced in animals and shown to block uptake of Tat protein in vitro [see, e.g., D. Brake et al, J. Virol., 64:962 (1990); D. Mann et al, EMBO J., 10:1733 (1991); J. Abraham et al, cited above; P. Auron et al, cited above; M. Jaye et al, cited above; G. Zauli et al, cited above]. More recent reports showed that monoclonal or polyclonal antibodies to Tat protein added to tissue culture medium attenuated HIV-1 infection in vitro [L. Steinaa et al, Arch. Virol., 139:263 (1994); M. Re et al, J. Acq. Imm. Def. Syndr. Hum. Retrovirol., 10:408 (1995); and G. Zauli et al, J. Acq. Imm. Def. Syndr. Hum. Retrovirol., 10:306 (1995)].
The inventor""s own publication [G. Goldstein, Nature Med., 2:960 (1996); see also, International Patent Application No. WO95/31999, published Nov. 30, 1995] reviewed the evidence indicating that secretion of HIV-1 Tat protein from infected cells and uptake by both infected and uninfected cells was important for the infectivity of HIV-1. Previous studies also showed that antibodies to Tat protein in vitro blocked uptake of Tat and inhibited in vitro infectivity. Goldstein proposed active immunization of mammals to induce antibodies to HIV-1 Tat protein as a potential AIDS vaccine.
Despite the growing knowledge about HIV-1 disease progression, there remains a need in the art for the development of compositions and methods for treatment of HIV-1, both prophylactically and therapeutically, which are useful to lower the viral levels of HIV-1 for the treatment and possible prevention of the subsequent, generally fatal, AIDS disease.
In one aspect, the invention provides as a novel composition comprising a peptide or polypeptide, which comprises an amino acid sequence selected from the formula referred to as Epitope I: R1-Val-Asp-Pro-Y-Leu-Glu-Pro-R2 [SEQ ID NO: 36], wherein Y is variously Arg, Lys, Ser or Asn. The N-terninal R1 may represent hydrogen (i.e., the hydrogen on the unmodified N terminal amino acid), or a lower alkyl, or a lower alkanoyl. R1 may also include a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl. In one embodiment, R1 is xe2x80x94X-Proxe2x80x94, wherein X is Glu or Asp. Preferably, R1 represents 2 amino acids. The C-terminal R2 can also represent the hydroxyl group on the C terminal amino acid or an amide. To enhance titer R2 is preferably a sequence of between 1 to about 14 additional amino acids amidated at the carboxyl terminus. In a preferred embodiment, R2 is xe2x80x94Trp-Lys-His-Pro-Gly-Serxe2x80x94 amide [SEQ ID NO: 10). The peptides or polypeptides of these compositions are produced synthetically or recombinantly. This composition may take the form of one or more of the above-described peptides expressed as a synthetic peptide coupled to a carrier, or expressed as a multiple antigenic peptide, or the selected peptides may be expressed within a recombinantly produced protein. This composition is designed to induce antibodies reactive with greater than 95% of the known variants of the HIV-1 Tat protein.
In another aspect, the above-described composition further contains one or more additional peptide or polypeptide(s) which represent other amino acid sequences which correspond to amino acid residues 2 or 4 to 10 of an HIV-1 Tat protein. These optional amino acid sequences are described in detail below. These sequences are preferably from an HIV-1 strain with a Tat protein variant at that location.
In another aspect, the invention provides a novel composition comprising a peptide or polypeptide of the formula referred to as Epitope II: R3-Lys-X-Leu-Gly-Ile-Ser-Tyr-Gly Arg-Lys-Lys-R4 [SEQ ID NO: 37]. According to this formula, X is Gly or Ala. The N terminal R3 may represent hydrogen (i.e., the hydrogen on the unmodified N terminal amino acid), or may be a lower alkyl, or a lower alkanoyl. R3 may also include a sequence of between 1 to about 5 amino acids, optionally substituted with a lower alkyl or lower alkanoyl. The C terminal R4 may be the free hydroxyl of the C terminal amino acid, or an amide, or a sequence of one or up to about 5 additional amino acids, optionally substituted with an amide. The peptides or polypeptides of these compositions are produced synthetically or recombinantly, provided that the recombinant Epitope II peptide is situated at the C terminus of the recombinant protein. This composition may take the form of one or more of the above-described peptides expressed as a synthetic peptide coupled to a carrier, or expressed as a multiple antigenic peptide. This composition is designed to induce antibodies reactive with greater than about 95% of the known variants of HIV-1 Tat protein.
In yet a further aspect, this invention provides a composition comprising a peptide or polypeptide of the formula referred to as Epitope III: R5-Arg-Arg-X-Z-A-Y-Ser-R6 [SEQ ID NO: 38], wherein X is selected from the group consisting of Ala, Pro, Ser and Gln; wherein Y is selected from the group consisting of Asp, Asn, Gly and Ser; wherein Z is selected from the group consisting of Pro and His; and wherein A is selected from the group consisting of Gln and Pro. The N terminal R5 is hydrogen, a lower alkyl, a lower alkanoyl, or a sequence of between 1 to about 3 amino acids, optionally substituted with a lower alkyl or lower alkanoyl. In a preferred embodiment R5 is xe2x80x94Gln-Argxe2x80x94, optionally modified as above. The C terminal R6 is either a free hydroxyl or an amide. A preferred embodiment of such a composition contains at least three Epitope III peptides, i.e., xe2x80x94Gln-Arg-Arg-Arg-Ala-Pro-Gln-Asp-Serxe2x80x94 (amino acids 54-62 of SEQ ID NO: 1), xe2x80x94Gln-Arg-Arg-Arg-Ala-His-Gln-Asp-Serxe2x80x94 (amino acids 2-10 of SEQ ID NO: 65), and xe2x80x94Gln-Arg-Arg-Arg-Ala-Pro-Pro-Asp-Serxe2x80x94 (amino acids 264-272 of SEQ ID NO: 3), optionally modified as above. Other peptides or polypeptides representative of amino acids 56-62 of Tat, but having different sequences from that of the above formula may also be included in the composition. The peptides or polypeptides of these compositions are produced synthetically or recombinantly. This composition may take the form of one or more of the above-described peptides expressed as a synthetic peptide coupled to a carrier, or expressed as a multiple antigenic peptide, or the selected peptides may be expressed within a recombinantly produced protein. This composition is designed to induce antibodies reactive with greater than about 75% of all known variants of HIV-1 Tat protein.
In still a further aspect, this invention provides a composition comprising a peptide or polypeptide of the formula referred to as Epitope IV: R7-Ser-Gln-X-His-Gln-Y-Ser-Leu-Ser-Lys-Gln-Pro-R8 [SEQ ID NO: 39], wherein X is selected from the group consisting of Asn and Thr; and wherein Y is selected from the group consisting of Ala and Val. The N terminal R7 may be hydrogen, a lower alkyl, a lower alkanoyl, or a sequence of between 1 to about 3 amino acids, optionally substituted with a lower alkyl or lower alkanoyl. The C terminal R8 may be a free hydroxyl, an amide, or a sequence of one or up to about 3 additional amino acids, optionally substituted with an amide. A preferred Epitope IV peptide is xe2x80x94Ser-Gln-Thr-His-Gln-Ala-Ser-Leu-Ser-Lys-Gln-Proxe2x80x94 [SEQ ID NO: 40]. The peptides or polypeptides of these compositions are produced synthetically or recombinantly. This composition may take the form of one or more of the above-described peptides expressed as a synthetic peptide coupled to a carrier, or expressed as a multiple antigenic peptide, or the selected peptides may be expressed within a recombinantly produced protein. This composition is designed to induce antibodies reactive with greater than 64% of all known variants of HIV-1 Tat protein.
In still another aspect, this invention provides composition described above that contains peptides or polypeptides which comprise one or more Epitope I peptides in combination with one or more Epitope II peptides, and/or one or more Epitope III peptides, and/or one or more Epitope IV peptides. Such compositions can combine appropriate Epitope peptides, so as to provide for a composition than induces antibodies reactive with greater than about 99% of all known HIV-1 Tat proteins.
In yet a further aspect, the invention provides a synthetic gene which encodes sequentially a peptide or polypeptide that contains at least one Epitope I amino acid sequence defined above, optionally with a carboxy terminal Epitope II peptide, or contains at least two Epitope I amino acid sequences. The synthetic gene may contain each amino acid sequence separated by a spacer sequence, or may express each peptide/polypeptide in an open reading frame with a carrier protein. The synthetic gene may be separated from the carrier protein by a spacer if the spacer is fused to an Epitope I sequence, leaving an Epitope II sequence at the carboxy terminus of the recombinant protein. Further embodiments include multiple Epitope I peptides fused together and to the carrier protein.
In yet a further aspect, the invention provides a synthetic molecule, e.g., a vector, comprising the above-described synthetic gene, operatively linked to regulatory nucleic acid sequences, which direct and control expression of the product of the synthetic gene in a host cell.
In another aspect, the invention provides a recombinant virus which contains the above described synthetic gene or synthetic molecule, which virus is capable of expressing multiple copies of the product of the gene or molecule in a host cell. The virus is non-pathogenic to humans.
In yet another aspect, the invention provides a commensal bacterium which contains the above described synthetic gene or synthetic molecule, which bacterium is capable of expressing multiple copies of the product of the gene or molecule and inducing antibodies in a mammalian host.
In still a further aspect, the invention provides an isolated antibody composition which is directed against a peptide or polypeptide of the compositions described above. Antibodies may also be obtained against multiple components of the compositions described above. This antibody is produced by immunizing a mammal with a peptide/polypeptide composition of the invention, a synthetic gene or synthetic molecule of the invention; a recombinant virus or commensal bacterium of the invention; and isolating and purifying antibody from said immunized mammal. Alternatively, the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or mixtures thereof.
Thus, another aspect of the invention is a pharmaceutical composition useful for inducing antibodies that react with greater than 95%, and preferably greater than 99%, of the known HIV-1 Tat proteins. These induced antibodies can impair the multiplication of HIV-1. The pharmaceutical composition comprises at least one of the recombinant or synthetic peptidelpolypeptide compositions described above; the synthetic gene/molecule described above; the recombinant virus described herein; or the commensal bacterium described herein, in a pharmaceutically acceptable carrier.
Still a further aspect of the invention is a pharmaceutical composition useful for impairing the multiplication of HIV-1, this composition containing an above described antibody composition.
In yet a further aspect of the invention, a method for reducing the viral levels of HIV-1 involves exposing a human to antibody-inducing pharmaceutical compositions described above, actively inducing antibodies that react with most HIV-1 Tat proteins, and impairing the multiplication of the virus in vivo. This method is appropriate for an HIV-1 infected subject with a competent immune system, or an uninfected or recently infected subject. The method induces antibodies which react with HIV-1 Tat proteins, which antibodies reduce viral multiplication during any initial acute infection with HIV-1 and minimize chronic viremia which leads to AIDS.
In still another aspect, the invention provides a method for reducing the viral levels of HIV-1 by administering to a human, who is incapable of mounting an effective or rapid immune response to infection with HIV-1, a pharmaceutical composition containing the antibody compositions described above. The method can involve chronically administering the composition.
Yet other aspects of the invention include methods for producing the compositions described above, as well as host cells transfected with such compositions.
Still another aspect of this invention is a kit useful for the measurement and detection of titers and specificities of antibodies induced by vaccination with the compositions described above. The kit of the invention includes peptides of Epitopes I through IV, and coated solid supports, a labelled reagent for detecting the binding of antibodies to these peptides, and miscellaneous substrates and apparatus for evoking or detecting the signals provided by the labels, as well as conventional apparatus for taking blood samples, appropriate vials and other diagnostic assay components.
In yet a further aspect, the invention provides a method for detecting the titers and reactivity patterns of antibodies in subjects vaccinated with the compositions of this invention. The method includes the steps of incubating dilutions of the subject""s biological fluid, e.g. serum, with plates or beads on which are bound one or more peptides of the Epitopes I through IV, washing away unbound biological materials, and measuring any antibody binding to the peptides with labeled reagent, e.g., an anti-human immunoglobulin to which is associated an enzyme. Depending on the type of label employed, the signal produced by the label may be evoked by further adding a substrate which reacts with the enzyme, e.g., producing a color change. Other conventional labels may also be incorporated into this assay design.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof